Water relaxation-based sensors

ABSTRACT

This invention relates to magnetic resonance-based sensors and related methods.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. ProvisionalApplication 60/679,437, filed on May 9, 2005, which is incorporatedherein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The work described herein was carried out, at least in part, using fundsfrom National Institutes of Health (NIH) Grants R01 EB004626 andEB00662. The government therefore has certain rights in the invention.

TECHNICAL FIELD

This invention relates to magnetic resonance-based sensors and relatedmethods.

BACKGROUND

Magnetic resonance (MR)-based reporting methods, such as magneticresonance imaging (MRI), offer certain known advantages as non-invasivemethods. For example, MRI can be used at tissue depths where opticalreporting methods can sometimes be complicated by light scattering andabsorption by the tissue, e.g., tissue depths greater than about 250 μm.

One application of nanotechnology in medicine is the development ofbiocompatible nanomaterials as environmentally sensitive sensors andmolecular imaging agents. Preparations of magnetic particles designedfor separation and extraction use particles that are amenable to easymanipulation by weak applied magnetic fields. These materials aretypically micron sized and have a high magnetic moment per particle.However, nanoparticles do not respond to the weak, magnetic fields ofhand held magnets.

SUMMARY

This invention relates generally to magnetic resonance-based sensors(e.g., water relaxation and equilibrium-based sensors) and relatedmethods, and is based, in part, on the discovery that sensors havingmagnetic nanoparticles encapsulated within a semipermeable enclosure canbe used as remote sensors for detecting various analytes in an aqueous,e.g., a water-containing, sample and can be used for the continuousmonitoring of changing levels of the analytes.

In its broadest aspects, the invention provides a water relaxation-basedsensor for detecting the presence of an analyte in a sample. The sensorincludes an enclosure defining an opening for entry of the analyte,e.g., a semipermeable membrane, and confined within said enclosure, aplurality of nanoparticles. The nanoparticles are suspended orsuspendable in an aqueous liquid phase, have a magnetic moment, e.g.,comprise crystalline iron oxide or other magnetic material, and arecovalently or non covalently linked to, or otherwise have immobilizedthereon, one or more moieties selected to alter the state of aggregationof the nanoparticles as a function of the presence or concentration ofthe analyte in the enclosure.

In one aspect, this invention features water relaxation-based sensorsfor detecting the presence of an analyte (e.g., an exogenous analyte) ina sample. The sensors include: (i) a walled enclosure enveloping achamber, wherein the wall includes one or more openings (e.g., a singleopening or a plurality of openings) for passage of the analyte into andout of the chamber; (ii) a plurality of magnetic nanoparticles locatedwithin the chamber, each nanoparticle having at least one moiety that iscovalently or noncovalently linked to (immobilized on) the nanoparticle;and optionally, (iii) at least one binding agent located within thechamber, in which the opening can be smaller in size than thenanoparticles and the binding agent and larger in size than the analyte;and the moiety and the analyte can each bind reversibly to the bindingagent, when present; or the analyte can bind reversibly to the moiety.In some embodiments, the openings can be larger than the binding agent,as long as the binding agents remain within the chamber when bound tothe nanoparticles.

Embodiments can include one or more of the following features.

The nanoparticles can be suspended or be suspendable in an aqueousliquid phase. The nanoparticles can have a magnetic moment generally, orunder certain conditions.

The wall can include more than one opening. When the wall contains morethan one opening (e.g., a plurality of openings), then at least one ofthe openings is smaller in size than the nanoparticles and optionallythe binding agent, and larger in size than the analyte. In certainembodiments, the wall includes a plurality of openings, in which each ofthe openings is smaller in size than the nanoparticles and the bindingagent, and each of the openings is larger in size than the analyte.

The moiety can be selected to alter the state of aggregation of thenanoparticles as a function of the presence or concentration of theanalyte in the enclosure.

In its several alternative embodiments, the sensor may exploit differentdetection formats. For example, the moiety may be selected to bind tothe analyte to produce an aggregate of plural linked nanoparticles as afunction of the presence or concentration of the analyte in theenclosure. The sensor may include an aggregate of plural linkednanoparticles, which is disaggregated as a function of the presence orconcentration of the analyte in the enclosure. The moiety may be afragment of an authentic sample of the analyte or a structural mimicthereof, in which case the sensor further includes a multivalent bindingagent, which binds to the analyte and the mimic (if used) to produce anaggregate of plural linked nanoparticles. The sensor can further includea multivalent binding agent which binds to the moiety to produce anaggregate. The sensor also can include a binding agent, which binds tothe moiety in the presence of the analyte to disassociate an aggregate.In yet another form, the sensor can include a binding agent that bindsto the moiety in the presence of the analyte to produce an aggregate. Inone embodiment, the sensor further includes a plurality of aggregatesconfined within the enclosure. In another embodiment, the sensorincludes a sample flow path in communication with the interior of theenclosure. Thus, the sample can flow into, or into and out of, theenclosure to permit periodic sampling of the analyte.

In some embodiments, the sensors include features (i), (ii), and (iii)above; the moiety can be, or can include as part of its chemicalstructure, a molecular fragment of the analyte being detected or amolecular fragment of a derivative, isostere, or mimic of the analytebeing detected; and the binding agent can be a protein. When the analyteis absent, the chamber can include one or more nanoparticle aggregates.Each of the nanoparticle aggregates can include one or morenanoparticles and the binding agent. Formation of the nanoparticleaggregate can occur through binding of a moiety on the nanoparticle tothe binding agent (e.g., the binding agent can include one or morebinding sites that are recognized for binding by the moiety).

When the analyte is present, the chamber can include substantiallydisaggregated nanoparticles. The analyte, when present, can displace thenanoparticles from the nanoparticle conjugates, thereby providingsubstantially disaggregated nanoparticles (e.g., the analyte and themoiety can be selected such that the analyte and the nanoparticles cancompete for binding with the binding agent, and the analyte, whenpresent, can displace the nanoparticles from the binding agent in theaggregate to provide disaggregated nanoparticles).

In certain embodiments, (a) when the analyte is absent, the chamber caninclude a nanoparticle aggregate, wherein the nanoparticle aggregate caninclude nanoparticles bound to the binding agent through the moiety; and(b) when the analyte is present, the nanoparticles are displaced fromthe binding agent by the analyte, and the chamber comprisessubstantially disaggregated nanoparticles. In the presence of awater-containing liquid media, the change in nanoparticle aggregation(from nanoparticle aggregates to substantially disaggregatednanoparticles and vice versa, e.g., the difference between (a) and (b)above) alters the proton relaxation of water inside of the chamber, butdoes not substantially alter the proton relaxation of water outside ofthe chamber.

In some embodiments, the sensors include features (i) and (ii) above,feature (iii) is absent; and the moiety can be, or can include as partof its chemical structure, a protein. When the analyte is absent, thechamber can include substantially disaggregated nanoparticles. When theanalyte is present, the chamber can include one or more nanoparticleaggregates. Each of the nanoparticle aggregates can include one or morenanoparticles and the analyte. Formation of the nanoparticle aggregatecan occur through binding of the analyte to the moiety on thenanoparticle (e.g., the moiety can include one or more binding sitesthat are recognized for binding by the analyte).

In certain embodiments, (a) when the analyte is absent, the chambercomprises substantially disaggregated nanoparticles; and (b) when theanalyte is present, the chamber comprises a nanoparticle aggregate,wherein the nanoparticle aggregate comprises nanoparticles bound to theanalyte through the moiety. In the presence of a water-containing liquidmedia, the change in nanoparticle aggregation (from nanoparticleaggregates to substantially disaggregated nanoparticles and vice versae.g., the difference between (a) and (b) above) alters the protonrelaxation of water inside of the chamber, but does not substantiallyalter the proton relaxation of water outside of the chamber.

In one aspect, this invention features methods of detecting an analytein an aqueous sample (e.g., monitoring the presence or concentration ofan analyte in a sample stream), the methods include: (i) providing asensor as described herein; (ii) measuring relaxation times (e.g., T2 orT1 relaxation times) of the water inside of the chamber of the sensor inthe absence of the analyte or under conditions that mimic the absence ofthe analyte; (iii) contacting the sensor with the sample (e.g., thenanoparticles can be suspended, or suspendable, in an aqueous liquidphase and can also have a magnetic moment); (iv) measuring relaxationtimes (e.g., T2 or T1 relaxation times) of the water inside of thechamber of the sensor; and (v) comparing the T2 relaxation timesmeasured in step (ii) and step (iv). A change (e.g., an increase ordecrease) in T2 relaxation times measured in step (iv) relative to theT2 relaxation times measured in step (ii) indicates the presence of theanalyte.

For example, one can flow a sample stream into the enclosure, allowanalyte in the sample to alter the state of aggregation of nanoparticles(e.g., suspended nanoparticles), and measures perturbation of themagnetic resonance relaxivity of water protons disposed adjacent thenanoparticles. These steps can be repeated to obtain a temporal profileof the concentration of the analyte in the stream.

Embodiments can include one or more of the following features.

The nanoparticles can be suspended or be suspendable in an aqueousliquid phase. The nanoparticles can have a magnetic moment.

The wall can include more than one opening. When the wall contains morethan one openings (e.g., a plurality of openings), then at least one ofthe opening is smaller in size than the nanoparticles and the bindingagent, and larger in size than the analyte. In certain embodiments, thewall includes a plurality of openings, in which each of the openings issmaller in size than the nanoparticles and the binding agent, and eachof the openings is larger in size than the analyte.

The moiety can be selected to alter the state of aggregation of thenanoparticles as a function of the presence or concentration of theanalyte in the enclosure. The moiety and the analyte can each bindreversibly to the binding agent, when present; or the analyte can bindreversibly to the moiety. The analyte can be a monovalent or multivalentanalyte.

The moiety can be, or can include as part of its structure, acarbohydrate, an antibody, an amino acid, a nucleic acid, anoligonucleotide, a therapeutic agent or a metabolite thereof, a peptide,or a protein. The moiety can be a covalently or noncovalently linkedanalyte (e.g., the analyte that is being detected, sometimes referred toas a bound analyte or a bound binding protein), a covalently ornoncovalently linked analyte derivative, or a covalently ornoncovalently linked analyte isostere or mimic (e.g., a derivative,isostere, or mimic of the analyte that is being detected).

In some embodiments, the moiety can be, or can include as part of itsstructure, a molecular fragment of the analyte being detected or amolecular fragment of a derivative, isostere, or mimic of the analytebeing detected.

As used herein and throughout, a moiety that includes “a molecularfragment of the analyte being detected” (or that includes a molecularfragment of a derivative, isostere, or mimic of the analyte beingdetected) is one in which a portion (e.g., a substantial portion) of thechemical structure of the analyte being detected (or a derivative,isostere, or mimic thereof) is incorporated into the chemical structureof the moiety. In these embodiments, the nanoparticle can have thegeneral formula (A): (A)_(z)-NP^(c); in which “A” is a molecularfragment of an analyte, A-X, in which X is a hydrogen atom or afunctional group that is present in the analyte, but not incorporatedinto the nanoparticle of formula (A); “NP^(c)” is the nanoparticle core,“-” is a covalent linkage (e.g., a chemical bond or linking functionalgroup) that connects any atom of the fragment to the nanoparticle; and zis 1-50 (e.g., 1-40, 1-30, 1-25, 1-20, 2-20). “A” in the above formulacan also be the molecular fragment of a derivative, isostere, or mimicof the analyte being detected.

The moiety can be a protein or a nucleic acid.

The binding agent can be absent. The moiety can be, or include as partof its structure, a protein. In some embodiments, (a) when the analyteis absent, the chamber includes substantially disaggregatednanoparticles; and (b) when the analyte is present, the chamber caninclude one or more nanoparticle aggregates. Each of the nanoparticleaggregates can include one or more nanoparticles and the analyte.Formation of the nanoparticle aggregate can occur through binding of theanalyte to the moiety on the nanoparticle (e.g., the moiety can includeone or more binding sites that are recognized for binding by theanalyte).

In some embodiments, (a) when the analyte is absent, the chambercomprises substantially disaggregated nanoparticles; and (b) when theanalyte is present, the chamber comprises a nanoparticle aggregate,wherein the nanoparticle aggregate comprises nanoparticles bound to theanalyte through the moiety.

The binding agent can be present, it can be, for example, a protein or amonoclonal antibody. The moiety can be, or can include as part of itsstructure, a molecular fragment of the analyte being detected or amolecular fragment of a derivative, isostere, or mimic of the analytebeing detected.

In some embodiments, (a) when the analyte is absent, the chamber caninclude one or more nanoparticle aggregates; and (b) when the analyte ispresent, the chamber can include substantially disaggregatednanoparticles. Each of the nanoparticle aggregates can include one ormore nanoparticles and the binding agent. Formation of the nanoparticleaggregate can occur through binding of a moiety on the nanoparticle tothe binding agent (e.g., the binding agent can include one or morebinding sites that are recognized for binding by a chemical group thatis present as all or part of the chemical structure of the moiety). Theanalyte, when present, can displace the nanoparticles from thenanoparticle conjugates, thereby providing substantially disaggregatednanoparticles (e.g., the analyte and the nanoparticle substituent can beselected such that the analyte and the nanoparticle moiety can competefor binding with the binding agent, and the analyte, when present, candisplace the nanoparticles from the binding agent in the aggregate toprovide disaggregated nanoparticles).

In some embodiments, (a) when the analyte is absent, the chamber caninclude a nanoparticle aggregate, wherein the nanoparticle aggregate caninclude nanoparticles bound to the binding agent through the moiety; and(b) when the analyte is present, the nanoparticles are displaced fromthe binding agent by the analyte, and the chamber comprisessubstantially disaggregated nanoparticles. The change in nanoparticleaggregation between (a) and (b) can alter the proton relaxation of waterinside of the chamber, but does not substantially alter the protonrelaxation of water outside of the chamber.

The change in nanoparticle aggregation between (a) and (b) can produce ameasurable change in the T2 relaxation times of water inside thechamber, and the change in the T2 relaxation times can be measurableusing a magnetic resonance imaging or non-imaging method.

The moiety can be linked to the nanoparticle by a functional group suchas —NH—, —NHC(O)—, —(O)CNH—, —NHC(O)(CH₂)_(n)C(O),—(O)C(CH₂)_(n)C(O)NH—, —NHC(O)(CH₂)_(n)C(O)NH—, —C(O)O—, —OC(O)—, or—SS—, in which n can be 0-20, e.g., 2, 5, 10, or 15. Each of theopenings can have a size (pore size) of from about 1 kDa to about 1 μm(e.g., about 1 kDa to about 300,000 kDa; about 1 kDa to about 100,000kDa; about 1 kDa to about 5 kDa; 1 kDa to about 3 kDa; 1 kDA to about 1μm).

Each of the nanoparticles can have a particle size or overall size offrom about 10 nm to about 500 nm (e.g., about 10 nm to about 60 nm,about 30 nm to about 60 nm). The overall size is the largest dimensionof a particle. The nanoparticle aggregate can have an overall size(particle size) of at least about 100 nm. The nanoparticles can besubstantially aggregated (e.g., include on or more nanoparticleaggregates) or substantially disaggregated. The analyte can be acarbohydrate (e.g., glucose).

The analyte can be chiral. The chiral analyte can be present togetherwith one or more optically active moieties in the sample. The chiralanalyte can be present together with a stereoisomer of the chiralanalyte in the sample. The chiral analyte can be present together withthe enantiomer of the chiral analyte in the sample. The chiral exogenousanalyte can be an amino acid.

The analyte can be a nucleic acid or an oligonucleotide.

The analyte can be a therapeutic agent, which as used herein refers to abioactive moiety, which when administered to a subject (e.g., a human oranimal subject) confers a therapeutic, biological, or pharmacologicaleffect (e.g., treats, controls, ameliorates, prevents, delays the onsetof, or reduces the risk of developing one or more diseases, disorders,or conditions or symptoms thereof) on the subject, or a metabolitethereof. The analyte can be, e.g., folic acid.

The analyte can be a peptide or a protein (e.g., influenza hemagglutininpeptide).

The moiety can be, or can include as part of its structure, a chiralmoiety. The moiety can be, or can include as part of its structure, anamino acid, a nucleic acid, an oligonucleotide, a therapeutic agent, ametabolite of a therapeutic agent, a peptide, or a protein. The moietycan be, or can include as part of its structure, a carbohydrate (e.g.,having the structure:

The binding agent can be a protein that includes at least two bindingsites or at least four binding sites. The binding agent can be arecombinant protein or be a complex of proteins each with binding sites.The complex of proteins may be assembled by crosslinking. The bindingagent can be a protein that binds to a carbohydrate (e.g., glucose). Theprotein can be conconavalin A. The binding agent can be a monoclonalantibody, a polyclonal antibody, or a oligonucleotide. The binding agentcan be, e.g., an antibody to folic acid or an antibody to influenzahemagglutinin peptide. The analyte and the nanoparticle can bindreversibly to the binding agent.

The magnetic nanoparticles each can include a magnetic metal oxide(e.g., a superparamagnetic metal oxide). The metal oxide can be ironoxide. Each of the magnetic nanoparticles can be an amino-derivatizedcross-linked iron oxide nanoparticle.

The sensor can be configured to be an implantable sensor. For example,the sensor can be implanted subcutaneously. In certain embodiments, thesensor can be implanted in an extremity of a subject (e.g., a human oranimal).

Steps (ii) and (iv) can include measuring T2 relaxation times or T1relaxation times. An increase in T2 relaxation times measured in step(iv) relative to the T2 relaxation times measured in step (ii) canindicate the presence of the analyte. A decrease in T2 relaxation timesmeasured in step (iv) relative to the T2 relaxation times measured instep (ii) can indicate the presence of the analyte.

The term “analyte” or “exogenous analyte” refers to a substance orchemical constituent (e.g., glucose, folic acid, or influenzahemagglutinin peptide) in a sample (e.g., a biological or industrialfluid) that can be analyzed (e.g., detected and quantified) andmonitored using the sensors described herein.

The term “subject” includes mice, rats, cows, sheep, pigs, rabbits,goats, horses, primates, dogs, cats, and humans.

A nanoparticle having at least one moiety described herein that iscovalently or noncovalently linked to the nanoparticle and that canswitch from being in an aggregated and disaggregated state is sometimesreferred to herein as a “magnetic nanoswitch” or “nanoswitch.”

Embodiments can have one or more of the following advantages.

While not wishing to be bound by theory, it is believed thatnanoparticle aggregation (formation of nanoparticle aggregates, e.g.,microaggregates) and disaggregation (formation of disaggregated ordispersed nanoparticles from microaggregates or nanoparticle agregates)is an equilibrium controlled process, and that the position of thisequilibrium is dependent upon (and therefore maintained by) analyteconcentration. When analyte concentration changes, the position of theequilibrium changes, at least in a range of sensitivity depending onseveral factors. This change in the position of this equilibrium ismanifested by changes in proton relaxation of the water inside of thesensor chamber, which is measurable. As such, the sensors have theadvantage of being useful for the continuous monitoring of changinglevels of analytes because there is generally no need to re-condition orreplace the sensors during the course of most ongoing (e.g., long term)measurements because essentially nothing is created or produced duringdetection; the equilibrium between aggregated and disaggregatednanoparticles is shifted by the analyte. As long as one can continuouslymonitor changes in the aforementioned equilibrium that occur inside thesensor chamber (e.g., by periodically or continuously monitoring T2relaxation times of the water inside of the chamber), then one cancontinuously monitor changing levels of analytes as those changes occur.

The sensors can be used to detect a chemically diverse array ofanalytes, which include without limitation, carbohydrates (e.g.,glucose), peptides (e.g., influenza hemagglutinin peptide), andtherapeutic agents (e.g., folic acid).

The sensors are relatively simple devices lacking moving parts,electronics and any connection to an outside recording device such as asampling tube or wire. Instead, the sensor operates by absorbing andemitting radiation at the Larmour precession frequency of water protons,which is interpretable as T2 and exogenous analyte (e.g., glucose)concentration. The radiation employed (e.g., 60 MHz for the 1.5 T MRI)penetrates biological systems, e.g., at depths where optical reportingmethods can sometimes be complicated by light scattering and absorptionby the tissue, e.g., tissue depths greater than about 250 μm. The sensorcan therefore be essentially a remote sensor, reporting on its localenvironment through water relaxation measurements while unconnected toan outside recording device or power source.

Analyte detection can take place in solution rather than on a surface,so as to avoid the need for developing and optimizing sensor surfacechemistry. This enables the features of an assay (sensitivity,specificity, kinetics) to be determined in a tube format, independentlyfrom the semi-permeable device or instrumentation needed to distinguishsensor water from bulk water. Binding agents, e.g., proteins andantibodies, and nanoparticles can readily be tested as reagents for newwater relaxation assays, leading a panel of relaxation-based sensors fordifferent analytes.

By sensing the position of the reversible equilibrium of nanoparticleaggregation/dispersion, the production or consumption of molecules isavoided as compared with an assay that includes irreversible reactions(e.g., single use assays). Thus, again there is no need to “recharge”the sensor with a substrate to continue operation. The sensors can beprepared for reuse, for example, by equilibrating in the absence of theanalyte or under conditions chosen to mimic the absence of the analyte(e.g., relatively low concentrations of the analyte).

The radiofrequency radiation used with the water relaxation sensorinteracts with water protons, rather than nanoparticles or biologicalmolecules, thereby minimizing the likelihood of radiation induceddamage.

The sensors are amenable for use in the detection of two or moreanalytes (e.g., a panel of different sensors each having, e.g., adifferent binding agent and moiety, can be used in the same screening ortesting environment to detect multiple analytes).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In case of conflict, thepresent application, including definitions will control. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

Although methods and materials similar or equivalent to those describedherein can be used in the practice of the present invention, preferredmethods and materials are described below. The materials, methods, andexamples are illustrative only and not intended to be limiting. Otherfeatures and advantages of the invention will be apparent from thedetailed description and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1A is a cross-sectional view of an embodiment of a waterrelaxation-based sensor for detecting a monovalent analyte. Also shownare the equilibrium controlled processes that occur in the sensorchamber in the absence and presence of the analyte and a summary of thewater relaxation properties of the aggregated and dispersednanoparticles.

FIG. 1B is a cross-sectional view of an embodiment of a waterrelaxation-based sensor for detecting a monovalent analyte. Also shownare the equilibrium controlled processes that occur in the sensorchamber in the absence and presence of the analyte and a summary of thewater relaxation properties of the aggregated and dispersednanoparticles.

FIG. 1C is a schematic representation of a sensor configurationembodiment in which the nanoparticles can bind directly to each other,and the analyte can mediate aggregation/disaggregation. The surface ofthis nanoparticle (left side of equation) is capable of binding to ananalyte and binding of the analyte can alter the physical properties ofthe surface, e.g., charge or hydrophobicity, resulting inaggregation/disaggregation (right side of equation). Here,aggregation/disaggregation is mediated by changes in pH (H+).

FIG. 1D is a schematic representation of a sensor configurationembodiment in which the detection of a sequence of bases on a nucleicacid fragment can mediate self-assembly of the nanoparticles. When thetwo types of nanoparticles are mixed, they can self-assemble via thehybridization that occurs between bases of the oligonucleotides (leftside of equation). An analyte that can bind to one of the types ofparticles can induce the dissociation of aggregates (right side ofequation).

FIG. 2 is a reaction scheme showing the synthesis of glucose linkedcross-linked iron oxide nanoparticle (Glu-CLIO).

FIG. 3A is a graphical representation of changes in T2 relaxation timesobtained in a tube based water relaxation assay for glucose using theGlu-CLIO/ConA configuration. Amino-CLIO does not react with ConA bindingprotein, as indicated by a stable T2 relaxation time after ConAaddition. Attachment of 2-amino-glucose (G) results in a functionalizednanoparticle, Glu-CLIO, which shows a T2 drop upon addition of aglucose-binding protein (ConA). The T2 drop is reversed by the additionof glucose. The data is indicative of nanoparticle aggregation anddisaggregation. For T2 measurements, 0.5 mL of Glu-CLIO (10 ug Fe/mL),800 ug/mL ConA in PBS with 1 mM CaCl₂ and 1 mM MgCl₂ were used.

FIG. 3B is a photograph of the assay apparatus. Iron concentration inthe photograph was increased to 100 ug Fe/mL for better contrast.

FIG. 4A is a graphical representation of changes in T2 relaxation timesobtained in a tube based assay for glucose using the Glu-CLIOnanoswitch/ConA configuration. Addition of ConA to Glu-CLIO caused ainitial T2 drop, which was reversed by the addition of increasingconcentrations of glucose. FIG. 4B is a graphical representation ofchanges in T2 values (at the plateau) obtained with different glucoseconcentrations. FIGS. 4C, 4D, and 4E are graphical representations ofparticle size distribution as obtained by light scattering experiments.FIG. 4C shows the particle size distribution for dispersed Glu-CLIOnanoparticles. FIG. 4D shows the switch from dispersed nanoparticles(dark bars) to the microaggregate state (light bars) upon ConA addition.FIG. 4E shows the switch of the Glu-CLIO nanoparticles back to thedispersed state upon glucose addition.

FIG. 5A is a graphical representation of changes in T2 relaxation timesobtained by contacting a water relaxation sensor with solutions ofvarying external glucose concentrations. Sensor was first placed in PBSwith 0.1 mg/mL glucose, then in buffer with 1.0 mg/mL glucose andreturned to a solution of 0.1 mg/mL glucose. Conditions were essentiallythe same as described with respect to FIGS. 3A and 3B.

FIG. 5B is a photograph of the sensor and the apparatus for containingthe glucose-containing sample media.

FIGS. 6A, 6B, and 6C are images corresponding to the reaction of a waterrelaxation sensor to glucose visualized by MRI. FIG. 5A shows sensorswith ConA and Glu-CLIO placed in 50 mL tubes. FIG. 5B shows an MR imageof a 50 mL tube with external glucose of 0.5 mg/mL FIG. 5C shows an MRimage of with an external glucose concentration of 1.4 mg/mL. Conditionswere essentially the same as described with respect to FIGS. 3A and 3B.

FIG. 7 is a graphical representation of time dependent changes in T2with increasing and decreasing glucose concentrations. The Glu-CLIOnanoswitch/ConA system used in the experiments described in FIGS. 4A-4Ewas placed in a semipermeable device so that glucose could be cycledbetween low and high concentrations, causing nanoparticles in the sensorto shift back and forth between a low T2 state and high T2 state.

FIG. 8A is a graphical representation of changes in T2 relaxation timesobtained in a tube based assay for influenza hemagglutinin peptide (HA)using the HA-CLIO nanoswitch/antibody to HA (anti-HA) configuration.Addition of anti-HA to HA-CLIO caused an initial T2 drop, which wasreversed by the addition of increasing concentrations of HA. FIG. 8B isa graphical representation of changes in T2 values obtained withdifferent HA concentrations. FIGS. 8C, 8D, and 8E are graphicalrepresentations of particle size distribution as obtained by lightscattering experiments. FIG. 8C shows the particle size distribution fordispersed HA-CLIO nanoparticles. FIG. 8D shows the switch from dispersednanoparticles (dark bars) to the microaggregate state (light bars) uponanti-HA addition. FIG. 8E shown the switch of the HA-CLIO nanoparticlesback to the dispersed state upon HA addition.

FIG. 9A is a graphical representation of changes in T2 relaxation timesobtained in a tube based assay for folic acid (FA) using the FA-CLIOnanoswitch/antibody to FA (anti-FA) configuration. Addition of anti-FAto FA-CLIO caused a initial T2 drop, which was reversed by the additionof increasing concentrations of FA. FIG. 9B is a graphicalrepresentation of changes in T2 values obtained with different FAconcentrations. FIGS. 9C, 9D, and 9E are graphical representations ofparticle size distribution as obtained by light scattering experiments.FIG. 9C shows the particle size distribution for dispersed FA-CLIOnanoparticles. FIG. 9D shows the switch from dispersed nanoparticles(dark bars) to the microaggregate state (light bars) upon anti-FAaddition. FIG. 9E shows the return of the FA-CLIO nanoparticles back tothe dispersed state upon FA addition.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

This invention relates generally to magnetic resonance-based sensors(e.g., water relaxation-based sensors) and methods for detecting variousanalytes (e.g., exogenous analytes) in water-containing media (e.g., invitro or in vivo media).

Sensors

In general, the sensors described herein include magnetic nanoparticles,or nanoparticles with a magnetic moment under certain conditions,encapsulated within a semipermeable walled enclosure, e.g., an enclosurethat retains the nanoparticles, but allows for passage of the analyteinto and out of the confines of the sensor chamber. The walled enclosurecan have one or more openings sized to enable the passage of theanalyte, but not the nanoparticles (and binding agent, when present).Each of the nanoparticles has at least one moiety (e.g., a molecularfragment of the analyte being detected or a molecular fragment of aderivative, isostere, or mimic of the analyte being detected; or aprotein) that is covalently or noncovalently linked to the nanoparticle.The sensor can further include a binding agent (e.g., a protein or amonoclonal antibody) also encapsulated within a semipermeable enclosure.The binding agent, when present, is capable of binding to the analyteand the moiety; and the moiety is capable of binding to the analyte orthe binding agent. In general, the moiety and the analyte can each bindreversibly to the binding agent, when present; or the analyte can bindreversibly to the moiety. Currently preferred chemistries for use in thepractice of the invention are described herein. Generally, the chemistryof the analyte, binding moiety, and binding agent, per se, unlessindicated otherwise herein, may be and typically is conventional, andmay be adapted from other arts for use in the novel sensors and methodsdisclosed herein.

Referring to FIG. 1A, in some embodiments, a water relaxation-basedsensor 10 for detecting a monovalent analyte (A^(ex) in FIG. 1A)includes a walled enclosure 12, a plurality of nanoparticles 20, and atleast one binding agent (e.g., a protein) 18. The walled enclosureencapsulates a chamber 16 and is perforated with a plurality of openings14. Both the nanoparticles 20 and the binding agent 18 are locatedwithin the confines of the chamber 16. Each of the nanoparticles 20 hasat least one moiety (A^(b) in FIG. 1A) that is covalently ornoncovalently linked to the nanoparticle and that includes a molecularfragment of the analyte being detected. The binding agent 18 is capableof binding (e.g., reversibly binding) to the analyte and the moietyA^(b). In all embodiments, the analyte is smaller in size than eitherthe nanoparticles 20 or the binding agent 18. In all embodiments, theopenings 14 are (i) larger in size than the analyte so as to allow theanalyte to pass freely into and out of the chamber 16 (arrows 17) and(ii) smaller in size than either the nanoparticles 20 or the bindingagent 18 so as to retain the nanoparticles 20 and the binding agent 18within the chamber 16.

When the analyte is absent, the nanoparticles 20 bind to the bindingagent 18 to form a nanoparticle aggregate 22 within the sensor chamber16. It is believed that binding of the nanoparticles 20 to the bindingagent 18 occurs through the moiety A^(b) (see FIG. 1A). In general,formation of the nanoparticle aggregate 22 is an equilibrium controlledprocess (arrows 23).

When the exogenous analyte is present and enters the chamber 16 throughopening 14, the binding agent-bound (e.g., binding protein-bound)nanoparticles of aggregate 22 are displaced from the binding agent bythe analyte (A^(ex) in FIG. 1A), thereby altering thenanoparticle-binding agent (binding protein) equilibrium (arrows 23). Asa result, a second equilibrium is established (arrows 25) in the chamber16 among the analyte, s analyte-binding agent (binding protein complex24, and (regenerated) nanoparticles 20. The regenerated nanoparticlesproduced in the second equilibrium controlled process (arrows 25) aresubstantially disaggregated relative to the bound nanoparticles ofaggregate 22 formed in the first equilibrium controlled process (arrows23).

Referring to FIG. 1B, in some embodiments, a water relaxation-basedsensor 10 for detecting a multivalent analyte (≡A^(ex) in FIG. 1B)includes a walled enclosure 12 as described elsewhere and a plurality ofnanoparticles 26. The nanoparticles are located within the confines ofthe chamber, and each of the nanoparticles has at least one moiety(e.g., at least one protein; at least 2, at least 3, at least 4) that islinked to the nanoparticle (hollow wedges in FIG. 1B). In allembodiments, the exogenous analyte is smaller in size than thenanoparticles 26. In all embodiments, the openings 14 are (i) larger insize than the exogenous analyte so as to allow the analyte to passfreely into and out of the chamber 16 (arrows 17) and (ii) smaller insize than the nanoparticles 26 so as to retain the nanoparticles 26within the chamber 16.

When the analyte is absent, the nanoparticles 26 are substantiallydisaggregated within the sensor chamber 16.

When the analyte is present and enters the chamber 16 through opening14, the nanoparticles 26 bind to the multivalent analyte (≡A^(ex) inFIG. 1B) to form a nanoparticle aggregate 28 within the chamber (arrows27). The nanoparticles that form part of aggregate 28 are substantiallyaggregated relative to nanoparticles 26. It is believed that binding ofthe nanoparticles 26 to the analyte occurs through the moiety (e.g., aprotein). In general, formation of the nanoparticle aggregate 28 is anequilibrium controlled process (arrows 27).

Referring to FIG. 1C, in some embodiments, the sensors can be configuredsuch that an analyte (e.g., a proton, H⁺) can directly mediateself-assembly (aggregation and disaggregation of the nanoparticles).Sensors having the configuration shown in FIG. 1C can have one or moreof the following properties: (i) the nanoparticles can bind directly toeach other (i.e., there are no molecules serving a “bridges” betweennanoparticles, as shown in FIGS. 1A and 1B); (ii) a single type ofnanoparticle can be employed, and (iii) an analyte can control theself-assembly by binding to the surface of the nanoparticle. In theseembodiments, the surface of the nanoparticle is capable of binding to ananalyte. Binding of the analyte can alter the physical properties of thesurface, e.g. charge or hydrophobicity. While not wishing to be bound bytheory, it is believed that the change in surface properties can alterthe attraction between the nanoparticles, and self-assembly (ordisassembly) of nanoparticles can occur. In certain embodiments, thesurface of the nanoparticle can be designed to have a surface that canbe charged or uncharged, as the pH is varied over some range ofinterest. For example, a peptide such asAc-LLLLLL-KHHHE-G-K(FITC)-C—NH₂, pI=6.48, can be attached tonanoparticles using bifunctional crosslinking agents such as SPDP orSIA, (see, e.g., Koch et al. “Uptake and metabolism of a dualfluorochrome Tat-nanoparticle in HeLa cells.” Bioconjug Chem. 2003;14(6): 1115). At a pH of about 6.48 or above, the histidine issubstantially unprotonated, and aggregation can occur throughself-association among the hydrophobic leucine side chains. At a pHbelow about 6.48, the histidine is substantially protonated, thenanoparticles carry a positive charge and are dispersed.

Referring to FIG. 1D, in some embodiments, the sensors can be configuredsuch that detection of a sequence of bases on a nucleic acid fragmentcan mediate self-assembly of the nanoparticles. Sensors having theconfiguration shown in FIG. 1D can have one or more of the followingproperties: (i) two types nanoparticles can be prepared which have anaffinity for each other, and (ii) one of the two types of nanoparticlesis capable of binding the analyte. In these embodiments, two types ofnanoparticles can be synthesized, each having a specific sequence ofsynthetic oligonucleotide attached. When the two types of nanoparticlesare mixed, they can self-assemble via the hybridization that occursbetween bases of the oligonucleotides. An example of suchdouble-stranded, oligonucleotide-mediated, nanoparticle aggregate isgiven in Perez et. al., “DNA-based magnetic nanoparticle assembly actsas a magnetic relaxation nanoswitch allowing screening of DNA-cleavingagents.” J Am Chem Soc. 2002; 124:2856. An analyte (e.g. a sequence ofbases present on a nucleic acid fragment) can then enter the sensor andby binding to one of the types of particles can induce the dissociationof aggregates.

Since the concentration dependent reaction of the analyte with thenanoparticle aggregate alters the nanoparticle aggregation state, thepresence and quantity of the exogenous analyte can be sensed, forexample, as a change in the T2 relaxation times of water inside of thesensor chamber. It is known, for example, that water T2 relaxation timesshorten upon aggregation or clustering of previously dispersed (e.g.,monodispersed, polydispersed) magnetic nanoparticles. While not wishingto be bound by theory, it is believed that during nanoparticleself-assembly into higher order nanoassemblies, the superparamagneticiron oxide core of individual nanoparticles becomes more efficient atdephasing the spins of the surrounding water protons (i.e., enhancingspin-spin relaxation times, e.g., T2 relaxation times).

Thus, in some embodiments, the analyte can be detected and quantified inthe sampling media by monitoring the relaxation properties of the waterthat is present within the sensor chamber 16 (e.g., measuring changes,e.g., increases and decreases, in T2 relaxation times of water that ispresent within the sensor chamber). For example, referring to FIG. 1A,the T2 relaxation times of the water inside of the sensor chamber 16 areexpected to decrease in the absence of analyte (due to formation of thenanoparticle aggregate 22) and then increase relative to these depressedvalues in the presence of analytes (due to displacement and subsequentdisaggregation of the bound nanoparticles of aggregate 22).Alternatively, referring to FIG. 1B, the T2 relaxation times of thewater inside of the sensor chamber 16 are expected to increase in theabsence of multivalent analyte and then decrease relative to thesevalues in the presence of the multivalent analytes. Since the bindingagent and/or the nanoparticles are confined within the chamber 16, thechanges in nanoparticle aggregation occurring within the sensor chamber16 in general do not substantially alter the proton relaxation of wateroutside of the chamber (i.e., bulk water).

Although measuring T2 can be a desirable method for determiningnanoparticle aggregation, any water relaxation phenomena associated withnanoparticles or with their change in aggregation state can be used. T2can generally be determined in a relatively fast and facile manner.However, measurements of nanoparticle aggregation can use T2 inconjunction with other relaxation processes such as T1. Measurements ofT1 and T2 can be used to correct for small changes in nanoparticleaggregation state within the sensor, due to a small expansion ofcontraction of the chamber. Accordingly, as used herein, references tomeasurement of relaxation phenomenon or magnetic relaxivity is intendedto embrace all such relaxation related processes, including measurementof TI.

Sensor Components and Specifications

In general, the size and shape of the sensor 10 can be selected asdesired.

In some embodiments, the sensors can be, for example, tubular,spherical, cylindrical, or oval shaped. The sensors described herein canhave other shapes as well.

In some embodiments, the size and shape of the sensor can be selected toaccommodate a desired or convenient sample holder size and/or samplevolume (e.g., in in vitro sensing applications). In general, the volumeof sensor can be selected to enable the sensor to distinguish betweenthe relaxation properties of water inside of the chamber and the wateroutside of the chamber. For example, the sensor size can be selected soas to accommodate a sample volume of from about 0.1 microliters (μL) toabout 1000 milliliters (mL) (e.g., about 1 μL (e.g., with animalimagers), 10 μL (e.g., with clinical MRI instruments) or 0.5 mL. Incertain embodiments, the sensor can have a tubular shape in which theopen end of the tube has a diameter of from about 1 millimeter (mm) toabout 10 mm (e.g., 5 mm 7.5 mm).

In some embodiments, the sensor size and shape can be selected on thebasis of the spatial resolution capabilities of conventional magneticresonance technology (e.g., in in vitro sensing applications). Incertain embodiments, the longest dimension of the sensor can be fromabout 0.01 mm to about 2 mm (e.g., 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1). In certain embodiments, the applied magnetic fieldcan be, for example, about 0.47 Tesla (T), 1.5 T, 3 T, or 9.4 T (animalassays generally).

The walled enclosure 12 separates the chamber 16 from the bulk samplemedia and provides one or more conduits (e.g., openings 14) for entry ofthe exogenous analyte (if present) from the bulk sample media. Ingeneral, the walled enclosure 12 can be any semipermeable material(e.g., a biocompatible semipermeable material) that is permeable to theexogenous analyte and water and substantially impermeable to thenanoparticles and the binding agent. In some embodiments, thesemipermeable material can be an ultrafiltration or dialysis membrane.In some embodiments, the semipermeable material can be a polymericsubstance (e.g., polymeric substances used for encapsulatingtransplanted cells, see, e.g., M. S. Lesney, Modern Drug Discovery 2001,4, 45). In some embodiments, the semipermeable material can be amaterial used in small implantable, sustained release devices (e.g.,those used in implantable, sustained release birth control devices,e.g., Depo-Provera, Norplant, Progestasert; or those described in C. I.Thompson et al., Can J Physiol Pharmacol 80, 180-92 (Mar, 2002) or D. C.Stoller, S. R. Thornton, F. L. Smith, Pharmacology 66, 11-8 (Sep,2002)).

In some embodiments, the walled enclosure is relatively resistant tofouling or coating under the sampling conditions, thereby increasinglythe likelihood that the walled enclosure can maintain the specified poresize of the openings 14 (e.g., increasing the likelihood that openings14 will remain substantially unblocked during sensing). Fouling is theclosure of pores (e.g., openings 14) due to the adsorption of proteinthat blocks pore. Fouling can be ascertained by placing materials inbiological fluids (e.g., blood) and evaluating their performance usingbiocompatibility testing methods known in the art.

In some embodiments, the walled enclosure 12 can be essentiallynonimmunogenic, thereby minimizing the likelihood of causing unwantedimmune or toxic side effects in a subject (e.g., a human).

Examples of biocompatible, semipermeable materials include withoutlimitation polysaccharide based materials (cellulose), modifiedcarbohydrate (cellulose ester), or polyvinyl pyrolidine.

In some embodiments, the walled enclosure 12 can be made of a relativelyinflexible semipermeable material, meaning that the encapsulated chamber16 is a true space or void that does not substantially change in volumewhen contacted with the fluid sample media. In other embodiments, thewalled enclosure can be a relatively flexible semipermeable material,meaning, for example, that the encapsulated chamber can expand in volumewhen contacted with the fluid sample media (e.g., by intake of the fluidsample media).

In general, the walls of the enclosure 12 are sufficiently thin to allowrapid sensor equilibration to changes in exogenous analyte levels. Insome embodiments, the membrane that forms the wall can have a thicknessof from about 1 and about 500 hundred microns.

In general, the pore size of the openings 14 can be selected so as tomeet the molecular exclusion criteria described herein (i.e., permeableto the exogenous analyte and water and substantially impermeable to thenanoparticles and the binding agent).

In some embodiments, molecular exclusion can be exclusion by molecularweight. In certain embodiments, each of the openings can have a poresize of from about 1 kDa to about 500,000 kDa (e.g., a pore size thatallows passage of molecules that have a certain molecular weight Each ofthe openings can have a size (pore size) of from about 1 kDa to about 1μm (e.g., about 1 kDa to about 300,000 kDa; about 1 kDa to about 100,000kDa; about 1 kDa to about 5 kDa; 1 kDa to about 3 kDa; 1 kDA to about 1μm). In certain embodiments, the openings can have a pore size of about1 kDa or about 3 kDa.

In certain embodiments, the semipermeable material can be Spectra/Por®tubing, Slide-A-Lyzer® microcassetes or dialysis fibers. Such materialsare generally preferred for applications not involving implantation. Ingeneral, the semipermeable material has a pore size that is larger insize than the analyte to permit passage of the analyte into and out ofthe chamber, but sufficiently small to retain magnetic nanoparticles andother reagents such as binding agents (e.g., a binding protein) withinthe confines of the chamber. The semipermeable material can be selectedfor the stability (long term function) in the fluid, which contains theanalyte to be measured (e.g., blood plasma, interstitial fluid, cerebralspinal fluid of a human or animal subject). The semipermeable materialcan be further selected on the basis of whether the sensor is implantedor whether the fluid to be assayed is contained within a vessel that isoutside of the subject (e.g., a bioreactor, tube or pipe).

The magnetic particles can be nanoparticles (e.g., having a particlesize of from about 10 nanometers (nm) to about 200 nm) or particles(e.g., having a particle size of from about 200 nm to about 5000 nm)provided that the particles remain essentially suspended (i.e., theparticles do not settle). As used herein, the term “magneticnanoparticles” refers to any particle that is always magnetic and anyparticle that has a magnetic moment under certain conditions (e.g., inan applied electromagnetic field). Particle settling can generally beavoided by using relatively small particles (e.g., nanoparticles) orrelatively large particles whose density is comparable to that of water.The density of particles can be altered by using polymers of differentdensities in their synthesis. In all embodiments, the nanoparticles orparticles have a surface that permits the attachment of biologicalmolecules. In some embodiments, the magnetic particles can benanoparticles having a particle size of from about 10 nm to about 500 nm(e.g., about 15 to about 200 nm, about 20 to about 100 nm, about 10 nmto about 60 nm, about 20 nm to about 40 nm, about 30 nm to about 60 nm,about 40 to 60 nm; or about 50 nm). The unfunctionalized metal oxidesare generally crystals of about 1-25 nm, e.g., about 3-10 nm, or about 5nm in diameter.

Magnetic materials larger than nanoparticles (particles) can be used. Ingeneral, such particles can have one or more of the followingproperties: (i) it is desirable that the particles have a relativelyhigh R2, i.e., alter water relaxation, (ii) it is desirable that theparticles not to have a high susceptibility to settle significantly bygravity during the time course of the assay, (iii) it is desirable thatthe particles have a surface for the attachment of biomolecules,preferably amino or carboxyl groups. Examples include microspheres offrom about 1-5 micron in diameter. Such particles can be obtained, e.g.,from commercial suppliers, which include Dynbead magnetic microspheresfrom Invitrogen (Carlsbad, Calif.), microspheres from Bangs Laboratories(Fishers, Ind.), and Estapor® Microspheres from Merck or EMD LifeSciences (Naperville, Ill.).

In some embodiments, the particles (e.g., nanoparticles 20 or 26) can beunfunctionalized magnetic metal oxides, such as superparamagnetic ironoxide. The magnetic metal oxide can also include cobalt, magnesium,zinc, or mixtures of these metals with iron. The term “magnetic” as usedherein means materials of high positive magnetic susceptibility such asparamagnetic or superparamagnetic compounds and magnetite, gamma ferricoxide, or metallic iron. In some embodiments, the nanoparticles 20include those having a relatively high relaxivity, i.e., strong effecton water relaxation.

In general, the particles can have a relatively high relaxivity owing tothe superparamagnetism of their iron or metal oxide. In someembodiments, the nanoparticles (e.g., 20 or 26) have an R1 relaxivitybetween about 5 and 30 mM⁻¹ sec⁻¹, e.g., 10, 15, 20, or 25 mM⁻¹ sec⁻¹.In some embodiments, the nanoparticles (e.g., 20 or 26) have an R2relaxivity between about 15 and 100 mM⁻¹ sec⁻¹, e.g., 25, 50, 75, or 90mM⁻¹ sec⁻¹. In some embodiments, nanoparticles (e.g., 20 or 26) have aratio of R2 to R1 of between 1.5 and 4, e.g., 2, 2.5, or 3. In someembodiments, the nanoparticles (e.g., 20 or 26) have an iron oxidecontent that is greater than about 10% of the total mass of theparticle, e.g., greater than 15, 20, 25 or 30 percent.

In some embodiments, when the magnetic nanoparticle is an ironoxide-based nanoparticle, concentrations of iron (Fe) can be from about2 micrograms (μg)/mL to about 50 μg/mL Fe. In general, the ironconcentration is selected so as to be sufficiently high to alter therelaxation properties of water. For particles with relatively highrelaxivities, lower iron concentrations can be used. For particles withrelatively low relaxivities, higher iron concentrations can be used.

Each of the nanoparticles (e.g., 20 or 26) includes at least one moiety(e.g., at least 2, at least 3, at least 4) that is covalently ornoncovalently linked to the nanoparticle.

In some embodiments, the moiety can be linked to the nanoparticle via afunctional group. The functional group can be chosen or designedprimarily on factors such as convenience of synthesis, lack of sterichindrance, and biodegradation properties. Suitable functional groups caninclude —O—, —S—, —SS—, —NH—, —NHC(O)—, —(O)CNH—, —NHC(O)(CH₂)_(n)C(O)—,—(O)C(CH₂)_(n)C(O)NH—, —NHC(O)(CH₂)_(n)C(O)NH—, —C(O)O—, —OC(O)—,—NHNH—, —C(O)S—, —SC(O)—, —OC(O)(CH₂)_(n)(O)—, —O(CH₂)_(n)C(O)O—,—OC(O)(CH₂)_(n)C(O)—, —C(O)(CH₂)_(n)C(O)O—, —C(O)(CH₂)_(n)C(O)—,—NH(CH₂)_(n)C(O)—, —C(O)(CH₂)_(n)NH—, —O(CH₂)_(n)C(O)—,—C(O)(CH₂)_(n)O—, —S(CH₂)_(n)C(O)—, —C(O)(CH₂)_(n)S—, —NH(CH₂)_(n)—,—(CH₂)_(n)NH—, —O(CH₂)_(n)—, —(CH₂)_(n)O—, —S(CH₂)_(n)—, or—(CH₂)_(n)S—, in which each n can be 1-100 (e.g., n can be 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99).Functional groups having cyclic, unsaturated, or cyclic unsaturatedgroups in place of the linear and fully saturated alkylene linkerportion, (CH₂)_(n), can also be used to attach the moiety to thenanoparticle. In certain embodiments, the functional group can beselected so as to render the nanoparticle larger in size than theopening(s) in the wall of the chamber so as to retain the nanoparticlewithin the confines of the chamber (e.g., where a relatively largeanalyte is being detected such as a lipoprotein).

In certain embodiments, the functional group can be—NHC(O)(CH₂)_(n)C(O)NH—, in which n can be 0-20. In certain embodiments,n can be 2, 3, 4, 5, or 6 (preferably, 2).

The functional group can be present on a starting material or syntheticintermediate that is associated with either the nanoparticle portion orthe moiety portion of the nanoparticles (e.g., 20 or 26).

In some embodiments, a nanoparticle-based starting material can containone or more functional groups for attachment of one or more moieties,(e.g., 2, 4, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45, or 50 functionalgroups). In certain embodiments, the nanoparticle can be anamino-derivatized cross-linked iron oxide nanoparticle (e.g., NH₂-CLIO).The number of moieties (e.g., a molecular fragment of the analyte beingdetected or a molecular fragment of a derivative, isostere, or mimic ofthe analyte being detected; or a protein) that are ultimately linked tothe nanoparticle can be equal to or less than the number of functionalgroups that are available for attachment of the moiety(ies) to thenanoparticle. In any event, it is permissible for the number of moietiesper nanoparticle to vary within a given population of nanoparticles(e.g., a population of 20 and/or 26).

In general, the number of moieties per nanoparticle can be selected asdesired (e.g., depending on the number and location of binding sites onthe binding agent (e.g., a protein) and/or if it is desired to have thenanoparticle cross link binding agents (e.g., proteins) when more thanone binding agent is present). In some embodiments, the magneticparticles can be multivalent particles in which multiple copies of amonovalent material are attached to the same particle. In general, thevalency can be from about 2.5 to about 20 copies of bound moiety pernanoparticle (i.e., average numbers of copies per nanoparticle, thussome particles can be monovalent within a given population of generallymultivalent particles). Higher levels are not necessarily believed to beneeded for function. Multivalent nanoparticles can be prepared byattaching two or more functional groups per nanoparticle. Multivalentnanoparticles can also be prepared by attaching either multivalent ormonovalent binding agents (e.g., proteins).

In general, moieties can include, without limitation, carbohydrates(e.g., glucose, polysaccharides), antibodies (e.g., monoclonalantibodies, biotinylated anti-GFP polyclonal antibody), amino acids aswell as derivatives and stereoisomers thereof (e.g. D-phenylalanine),chiral moieties, lipids, sterols, lipopolysaccharides, lipoproteins,nucleic acids, oligonucleotides, therapeutic agents (e.g., folic acid),metabolites of therapeutic agents, peptides (e.g., influenzahemagglutinin peptide), or proteins.

In some embodiments, the moiety can be, or include as part of itschemical structure, a molecular fragment of the analyte being detectedor a molecular fragment of a derivative, isostere, or mimic of theanalyte being detected.

In general, such a moiety is one that is (i) recognized by the bindingagent (e.g., protein, e.g., a binding protein) and (ii) displaceablefrom the binding agent by the analyte (i.e., the analyte can competewith the moiety for binding to the binding agent (e.g., a protein)).Thus, in some embodiments, the moiety and the analyte being detected canbe substantially similar in structure to one another and havesubstantially similar binding affinities towards the binding agent. Inother embodiments (such as when the moiety is, or includes as part ofits chemical structure, a molecular fragment of a derivative, isostere,or mimic of the analyte being detected), the moiety and the analytebeing detected may not necessarily be substantially similar instructure, but may have substantially similar binding affinities towardsthe binding agent.

Nanoparticles having at least one moiety that is a molecular fragment ofthe analyte being detected or a molecular fragment of a derivative,isostere, or mimic of the analyte being detected can have the generalformula (A):(A)_(z)-NP^(c)  (A)

in which:

moiety “A” is a molecular fragment of an analyte (or a derivative,isostere, or mimic thereof), A-X, wherein X is a hydrogen atom or afunctional group that is present in the free analyte (or a derivative,isostere, or mimic thereof), but not incorporated into the nanoparticleof formula (A);

“NP^(c)” is the nanoparticle core;

“-” is a covalent linkage (e.g., a chemical bond or any linkingfunctional group described herein) that connects any atom of thefragment to the nanoparticle; and

z is 1-50 (e.g., 1-40, 1-30, 1-25, 1-20, 2-20, 2, 4, 6, 8, 10, and 15).

In certain embodiments, X can be a hydrogen atom that forms part of anamino or hydroxy group that is present in the analyte; or X can befunctional group, such as an amino group or a hydroxy group. By way ofexample, for a given analyte A-OH (X=OH=hydroxy group), thecorresponding nanoparticle can have, for example and without limitation,the structure (A-O)_(z)-NP^(c) or (A-NH)_(z)-NP^(c).

In some embodiments, the moiety can include a carbohydrate as part ofits chemical structure (e.g., glucosyl). In certain embodiments, themoiety can include a molecular fragment of a carbohydrate analyte beingdetected or a molecular fragment of a derivative, isostere, or mimic ofa carbohydrate analyte being detected. For example, the moiety can haveformula (I):

in which the wavy line indicates the point of connection of the moietyto the nanoparticle. The moiety of formula (I) can be used inconjunction with a sensor for detecting and quantifying glucose.

The moiety can be a monovalent or multivalent protein (e.g., having atleast two (e.g., three, four, five, or six)) binding sites.

In certain embodiments, the nanoparticle can further include asubstituent that serves to render the nanoparticle larger in size thanthe opening(s) in the wall of the chamber so as to retain thenanoparticle within the confines of the chamber (e.g., where arelatively large analyte is being detected such as a lipoprotein).

In some embodiments, the binding agent can be absent (e.g., whendetecting multivalent analytes with nanoparticles in which thecovalently or noncovalently linked moiety is, e.g., a protein; see,e.g., assay configuration delineated in FIG. 1B). Thus the assay canmeasure multivalent analyte proteins, using a sensor with a pore sizethat is large enough to allow analyte to enter and leave the chamber,while retaining nanoparticles, i.e. the pore size in FIG. 1A and FIG.1B. can be adjusted. Various assay configurations are described herein.

In some embodiments, the binding agent can be present (e.g., whendetecting monovalent analytes with nanoparticles, which include amolecular fragment of the analyte being detected or a molecular fragmentof a derivative, isostere, or mimic of the analyte being detected; see,e.g., assay configuration delineated in FIG. 1A). The binding agent canbe, for example, a protein, an antibody, a lectin, a receptor bindingprotein, a binding domain of a protein, a synthetic material, or anon-protein material.

In some embodiments, the binding agent can be a protein. In certainembodiments, the binding protein can be a multi-valent binding proteinhaving at least two binding sites (e.g., three, four, five, or six).

In general, binding between the binding agent (e.g., a protein orantibody) and the nanoparticle (via the moiety) and the analyte isreversible; and binding between a moiety and an analyte is reversible.As such, the analyte can be detected and quantified in a non-consumptivemanner (i.e., the binding agent or the moiety reversibly binds, but doesnot consume, the analyte). This reversibility provides a steady statecondition for bound and unbound analyte that can be quantitated. Analyteconcentration can then be mathematically calculated using conventionalmethods. A person of ordinary skill in the art would recognize that, forexample, the reaction kinetics associated with binding and release ofthe analyte can be different for each protein selected as a bindingagent.

In certain embodiments, the protein binding agent can bind a therapeuticagent or metabolite thereof; a carbohydrate (e.g., glucose; e.g., theprotein can be conconavalin A); or an amino acid (e.g., the bindingprotein can selectively bind one enantiomer over another, e.g.,D-alanine versus L-alanine). In certain embodiments, the protein can begreen fluorescent protein (GFP).

In certain embodiments, the protein binding agent can be a monomer. Inother embodiments, the protein binding agent can be multimeric bindingagent (e.g., prepared by making a fusion protein that includes severalcopies of one protein or cross-linking monomers to create a multivalentbinding moiety). In still other embodiments, the binding agent can be anenzyme, which is modified so that it can bind to a substrate, but doesnot catalyze a reaction.

In certain embodiments, the binding agent can bind a lipid, a sterol, alipopolysaccharide, or a lipoprotein. For example, the binding agent canbe a protein that binds a sterol (e.g., apoSAAp for binding cholesterol,see, e.g., Liang and Sipe, 1995, “Recombinant human serum amyloid A(apoSAAp) binds cholesterol and modulates cholesterol flux”, J. LipidRes, 36(1): 37). As another example, the binding agent can be a receptorthat binds a lipoprotein (e.g., a soluble low density lipoprotein and/ormutants thereof, see, e.g., Bajari et al, 2005, “LDL receptor family:isolation, production, and ligand binding analysis”, Methods,36:109-116; or Yamamoto et al., 2005, “Characterization of low densitylipoproteinreceptor ligand interactions by fluorescence resonance energytransfer”, J. Lipid Research, 47:1091. As a further example, the bindingagent can be a protein that binds a fatty acid (e.g., human serumalbumin, see, e.g., Fang et al, 2006, “Structural changes accompanyinghuman serum albumin's binding of fatty acids are concerted”,1764(2):285-91. Epub Dec. 27, 2005).

In other embodiments, the binding agent can be a monoclonal antibody, apolyclonal antibody, or a oligonucleotide. The binding agent can be,e.g., an antibody to folic acid or an antibody to influenzahemagglutinin peptide.

In some embodiments, the analyte can be chiral. The chiral analyte canbe present together with one or more optically active moieties in thesample (e.g., a stereoisomer of the chiral analyte in the sample, e.g.,the enantiomer of the chiral analyte in the sample). In certainembodiments, the chiral analyte can be an amino acid.

The sensors described herein can be used to detect, monitor, andquantify a of analytes that can include, without limitation, ions, smallmolecules, proteins, viruses and lipoproteins (see Table 1). TABLE 1Analytes that can be measured by water relaxation sensors Type ofanalyte Analyte (size in kDa unless otherwise noted) T4, T3, cortisolSmall molecule hormone (<1) Thyroid stimulating hormone (TSH), Proteinhormones (10-100) human chorionic gonadotropin (hCG), leutinizinghormone (LH), follicle stimulating hormone (FSH) Troponin, C-reactiveprotein (CRP), Proteins for inflammation or Creatine phosphokinase(CPK-MB, CPK- heart attack (10-100) BB), myoglobin Prostatic specificantigen (PSA), Proteins for cancer detection (10-100) Carcinomaembryonic antigen (CEA), alphafetoprotein (AFP) Low density lipoprotein(LDL), high Lipoproteins for lipid status density lipoprotine (HDL)(500-2000) Ferritin Protein for iron anemia (400-600) Paclitaxel Smallmolecule cancer chemotherapeutic (<1) B12/Folate Small moleculenutrients and cofactors Theophyline, gentamycin, tobramycin, Therapeuticdrug (<1) valproate Digoxin, digitoxin Therapeutic drug (<1) GlucoseGlucose (<1) Hydrogen ions Metabolite (<1) Calcium ion Metabolite (<1)Herpes simplex virus Virus (1 μm) Human immunodeficiency virus Virus (1μm) (HIV) Hepatitis A, B or C Virus (1 μm)

In certain embodiments, the analyte can be a carbohydrate (e.g.,glucose); a lipid, a sterol, a lipopolysaccharide, a lipoprotein, anucleic acid or an oligonucleotide; therapeutic agents (e.g., folicacid), metabolites of therapeutic agents, peptides (e.g., influenzahemagglutinin peptide), or a protein.

Sensor Manufacture and Use

In some embodiments, nanoparticles having reactive functional groups,(e.g., electrophilic functional groups such as carboxy groups ornucleophilic groups such as amino groups) can be employed as startingmaterials for the nanoparticles used in conjunction with the sensors.

Carboxy functionalized nanoparticles can be made, for example, accordingto the method of Gorman (see WO 00/61191). In this method, reducedcarboxymethyl (CM) dextran is synthesized from commercial dextran. TheCM-dextran and iron salts are mixed together and are then neutralizedwith ammonium hydroxide. The resulting carboxy functionalizednanoparticles can be used for coupling amino functionalized groups,(e.g., a further segment of the functional group or the substratemoiety).

Carboxy-functionalized nanoparticles can also be made frompolysaccharide coated nanoparticles by reaction with bromo orchloroacetic acid in strong base to attach carboxyl groups. In addition,carboxy-functionalized particles can be made from amino-functionalizednanoparticles by converting amino to carboxy groups by the use ofreagents such as succinic anhydride or maleic anhydride.

Nanoparticle size can be controlled by adjusting reaction conditions,for example, by using low temperature during the neutralization of ironsalts with a base as described in U.S. Pat. No. 5,262,176. Uniformparticle size materials can also be made by fractionating the particlesusing centrifugation, ultrafiltration, or gel filtration, as described,for example in U.S. Pat. No. 5,492,814.

Nanoparticles can also be synthesized according to the method of Molday(Molday, R. S. and D. MacKenzie, “Immunospecific ferromagneticiron-dextran reagents for the labeling and magnetic separation ofcells,” J. Immunol. Methods, 1982, 52(3):353-67, and treated withperiodate to form aldehyde groups. The aldehyde-containing nanoparticlescan then be reacted with a diamine (e.g., ethylene diamine orhexanediamine), which will form a Schiff base, followed by reductionwith sodium borohydride or sodium cyanoborohydride.

Dextran-coated nanoparticles can be made and cross-linked withepichlorohydrin. The addition of ammonia will react with epoxy groups togenerate amine groups, see, e.g., Josephson et al., Angewandte Chemie,International Edition 40, 3204-3206 (2001); Hogemann et al., Bioconjug.Chem., 2000, 11(6):941-6; and Josephson et al., “High-efficiencyintracellular magnetic labeling with novel superparamagnetic-Tat peptideconjugates,” Bioconjug. Chem., 1999, 10(2):186-91. This material isknown as cross-linked iron oxide or “CLIO” and when functionalized withamine is referred to as amine-CLIO or NH₂-CLIO.

Carboxy-functionalized nanoparticles can be converted toamino-functionalized magnetic particles by the use of water-solublecarbodiimides and diamines such as ethylene diamine or hexane diamine.

Nanoparticles 20 having a moiety corresponding to formula (I) can beprepared by contacting amino-CLIO (NH₂-CLIO) with succinic anhydride (pH8.5) followed by 2-aminoglucose in the presence of a carbodiimide (e.g.a water soluble carbodiimide, e.g.,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride(EDC)/N-hydroxysuccinimide (NHS), pH 6.0 (see FIG. 2). Suchnanoparticles are referred to herein as “G-CLIO,” “Glu-CLIO,” or“Glu-CLIO nanoswitches.”

Folic acid (FA) can be conjugated to NH₂-CLIO using a water solublecarbodiimide (e.g. EDC/NHS, pH 6.0) to provide nanoparticles 20 having afolic acid-containing moiety linked to the nanoparticle. Suchnanoparticles are referred to herein as “FA-CLIO” or “FA-CLIOnanoswitches.”

Influenza hemagglutinin peptide (HA) can be conjugated to NH₂-CLIO with,e.g., N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) (PBS buffer,pH 7.4 to provide nanoparticles 20 having an HA-containing moiety linkedto the nanoparticle. Such nanoparticles are referred to herein as“HA-CLIO” or “HA-CLIO nanoswitches.”

The nanoparticles used in the sensors described herein can also beprepared using the conjugation chemistry described in, e.g., Sun, E. Y.,Josephson, L., Kelly, K., Weissleder, R. Bioconjugate Chemistry 2006,17, 109-113, which is incorporated by reference herein. Thenanoparticles used in the sensors described herein can also be preparedusing “click chemistry” methodology described in, e.g., Kolb et al,Angew Chem Int Ed Engl., 2001, 40:2004-2021.

The combination of nanoparticles 20, analyte, and protein binding agent18 used in the sensors described herein can be selected as desired.

In some embodiments, the combination of nanoparticles, analyte, andbinding agent used in the sensors can be based on the assayconfigurations described in, e.g., Josephson, et al., Angewandte Chemie,International Edition 40, 3204-3206 (2001); Perez et al., Nat Biotechnol20, 816-20 (2002); J. M. Perez et al. J Am Chem Soc 124, 2856-7 (2002);and Tsourkas et al. Angew Chem Int Ed Engl 43, 2395-9 (2004) (a waterrelaxation assay using antibodies and surface functionalizednanoparticles detecting enantiomeric impurities indicating the abilityof the MR-based assay to measure the levels of drugs or metabolitesrather than glucose), each of which is incorporated by reference herein.

By way of example, Table 2 illustrates representative assayconfigurations. TABLE 2 Relaxation Sensor Assay Type or Example andConfiguration Exogenous Analyte Binding Agent Comment See, e.g., FIG. 1Aglucose conA See Examples See, e.g., FIG. 1A Chiral Moieties (e.g.,Monoclonal See, e.g., All Figures Small molecule antibodies of Tsourkas,A., Enantiomers) Hofstetter, O., Hofstetter, H., Weissleder, R., andJosephson, L. (2004). Magnetic relaxation switch immunosensors detectenantiomeric impurities. Angew Chem Int Ed Engl 43, 2395-2399. See,e.g., FIG. 1A Monovalent analytes Multivalent binding Multivalent (e.g.,therapeutic agent (e.g., able to Functionalized agents, peptides, bindtwo or more nanoparticle must be nucleic acids, etc.) nanoparticles(able to two binding simultaneously; e.g., proteins Lectin, polyclonalor simultaneously) monoclonal antibody) See, e.g., FIG. 1B Nucleic acidoligonucleotide See e.g., all figures of Josephson, L., Perez, J. M.,and Weissleder, R. (2001). Magnetic nanosensors for the detection ofoligonucleotide sequences. Angewandte Chemie, International Edition 40,3204-3206. See, e.g., FIG. 1B mRNA oligonucleotide E.g., FIG. 4 ofPerez, J. M., Josephson, L., O'Loughlin, T., Hogemann, D., andWeissleder, R. (2002). Magnetic relaxation switches capable of sensingmolecular interactions. Nat Biotechnol 20, 816-820 See, e.g., FIG. 1BGFP Polyclonal antibodies E.g., FIG. 5A, Perez, J. M., Josephson, L.,O'Loughlin, T., Hogemann, D., and Weissleder, R. (2002). Magneticrelaxation switches capable of sensing molecular interactions. NatBiotechnol 20, 816-820. See, e.g., FIG. 1B Proteins, Proteins,Multivalent polysaccharide Exogenous Analyte (able to simultaneouslybind two or more nanoparticles). Multivalent functionalized nanoparticle(able to bind to two or more analytes simultaneously.)

In some embodiments, the sensors described herein can be used to monitorphysiological concentrations of glucose (see Examples section).

In general, any MR-based method that is capable of discriminating waterT2 relaxation times in the sensor chamber from those outside of thesensor chamber can be used to monitor the sensors. Such methods can beMR imaging or MR non-imaging methods.

For example, in applications where a single water relaxation sensor isemployed, and the T2 of non-sensor, bulk water is uniform, determiningthe relaxation properties of sensor water may not necessarily require anMR imager or a two-dimensional matrix of water relaxation data. Anynon-imaging method capable of distinguishing the properties of the MRsignal emanating from inside the sensor from those of the bulk watercould be used. Thus, far simpler and less costly types ofinstrumentation than a clinical MRI instrument can distinguish therelaxation properties sensor water from bulk water. First, the sensorcan be implanted in a tube of flowing tube of fluid, minimizing thevolume of a homogeneous magnetic field needed but still using thespatial encoding methods of MRI instrumentation. Second, the appliedmagnetic field need not be homogeneous, a requirement of magnets used togenerate MR images. Selective excitation magnets were considered inearly MR imager designs (see, e.g., Z. Abe, K. Tanaka, Y. Yamada, RadiatMed 2, 1-23). A variable field strength hand-held magnet andexcitation/receiver coil are used for analyzing the relaxationproperties of samples within several millimeters of the magnet incommercial devices, see, e.g.,http://www.minispec.com/products/ProFiler.htm. These devices can be usedwith the new sensors.

Solvent, (e.g., water), spin-spin relaxation times (T2) can bedetermined by relaxation measurements using a nuclear magnetic resonancebenchtop relaxometer. In general, T2 relaxation time measurements can becarried out at 0.47 T and 40° C. (Bruker NMR Minispec, Billerica, Mass.)using solutions with a total iron content of 10 μg Fe/mL.

Alternatively, T2 relaxation times can be determined by magneticresonance imaging of 384-well plates (50 μL sample volume), allowingparallel measurements at higher throughput. In general, magneticresonance imaging can be carried out using a 1.5 T superconductingmagnet (Sigma 5.0; GE medical Systems, Milwaukee, Wis.) usingT2-weighted spin echo sequences with variable echo times (TE=25-1000 ms)and repetition times (TR) of 3,000 ms to cover the spectrum of theanticipated T2 values. This technique is described in, for example,Perez, J. M., et al. Nat Biotechnol 2002, 20, 816-820; and Hogemann, D.,et al. Bioconjug Chem 2002, 13, 116-121.

While not wishing to be bound by theory, it is believed thatnanoparticle aggregation is associated with an increase in R2 relaxivityof nanoparticles, but not necessarily with R1 relaxivity. See, e.g.,Table 1 Josephson, L., Perez, J. M., and Weissleder, R. (2001).“Magnetic nanosensors for the detection of oligonucleotide sequences.”Angewandte Chemie International Edition: 3204-3206. Relaxivity, R=changein relaxation rate (1/T) per change in concentration by 1 mM. Sincethere is essentially no change or relatively little change in R1associated with nanoparticle aggregation, the measurement of T1 can beused to determine nanoparticle concentration, while measurements of T2can be used to determine nanoparticle aggregation. This method can useby way of example equations (1)-(3) below, where NP=nanoparticle, andT1_(o) and T2_(o) are the relaxation times of sensor water in theabsence of nanoparticles. [A], the concentration of analyte, can be asimple or complex function of R2, which reflects the aggregation stateof the particles.[NP]=(1/T1_(o)−1/T1_(NP))/R1  (1)(1/T2_(o)−1/T2_(NP))/[NP]=R2  (2)[A]=kR2 or [A]=f(R2)  (3)

Nanoparticle aggregation can also be determined without measurement ofT2 as the examples below indicate.

1. Measurement of the T2*, or free induction decay, rather than T2.

2. Measurement of amount of relaxation properties of specific class ofwater protons in the sample using an off resonance radiation, that isradiation that is not precisely at the Larmour precession frequency. Inthis measure a frequency of incident radiation not precisely at theLarmour precession frequency is employed.

3. Measurement of the height of a single echo obtained with a T2measuring pulse sequence rather than a complete echo train. Normal T2measurements utilize the declining height of a number of echoes todetermine T2.

4. Shifting the frequency or strength of the applied magnetic field,measuring the broadness of the proton absorption peak. Broader the peaksor energy absorption are correlated with higher values of T2.

In some embodiments, with instrumentation designed solely fordistinguishing the relaxation of sensor water protons from bulk waterprotons, relaxation based sensors can be used to monitor exogenousanalytes in any enclosed, aqueous system including bioreactors or fluidsin a variety of industrial applications.

In some embodiments, the sensor can be an implantable sensor implantedsubcutaneously (e.g., the sensor can be implanted in an extremity of asubject so as to avoid having the entire body of the subject surroundedby the magnetic field).

EXAMPLES

The invention is further illustrated by the following Examples. TheExamples are provided for illustrative purposes only, and are not to beconstrued as limiting the scope or content of the invention in any way.

General

Synthesis of surface functionalized nanoparticles: EDC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), sulfo-NHS(sulfosuccinimidyl ester of N-hydroxysuccinimide) were purchased fromPierce. SPDP (N-succinimidyl 3-(2-pyridyldithio) propionate) waspurchased from Molecular Biosciences. All other chemicals were purchasedfrom Sigma Aldrich and used as received. Amino-CLIO nanoparticles weresynthesized by crosslinking the dextran coating with epichlorohydrin andreacting it with ammonia, to provide primary amine groups (see, e.g.,Josephson, L.; Tung, C. H.; Moore, A.; Weissleder, R. Bioconjug Chem.1999, 10, (2), 186-91.; and Josephson, L.; Perez, J. M.; Weissleder, R.Angewandte Chemie, International Edition 2001, 40, (17), 3204-3206). Thenumber of amines was determined by reaction with SPDP and treatment withdithiothreitol that releases pyridine-2-thione (P2T) (see, e.g., Zhao,M.; Kircher, M. F.; Josephson, L.; Weissleder, R. Bioconjug Chem 2002,13, (4), 840-4). Protein binding agents ConA, anti-folate acid antibody(anti-FA) and anti-HA antibody (anti-HA) were purchased from Sigma.

For T2 measurements, a 0.47T relaxometer (Bruker) was used. Measurementswere made in 0.5 mL of PBS at 40° C. The concentration of surfacefunctionalized nanoparticles was between 8 and 15 μg/ml Fe, adjusted togive a starting T2 of about 150 msec. The concentrations of bindingproteins were 1 mg/mL (Con A), 0.1 mg/mL (anti-HA) and 0.1 mg/mL(anti-FA). After addition of each amount of analyte, T2 was recordedseveral times until it reached a stable value.

The size of nanoparticles was measured by a laser light scattering(Zetasizer, Malvern Instruments) in 1 mL PBS at 23° C. and is thevolume-based size. The concentration of surface functionalizednanoparticles was between 25 and 35 μg/ml Fe, which is believed to beoptimal for obtaining nanoswitch size distribution on this instrument.The concentrations of binding proteins were 1 mg/mL (Con A), 0.5 mg/mL(anti-HA) and 0.5 mg/mL (anti-FA). Repeated size measurements were madeat 40-80 minutes post addition of binding protein or analyte and wereessentially constant over than period. Results shown are typical sizedistributions. For reversion to the dispersed states (FIGS. 4E, 8E and9E), 600 mg/dl glucose, 500 nM HA and 30 nM FA were employed.

For some of the experiments enclosing the nanoswitches in asemipermeable device, 0.25 ml of Glu-CLIO (10 μg/mL Fe) and ConA (1mg/mL) were placed in membrane with a 10 kDa cutoff (Spectra/Por,Fischer). The device was transferred back and forth between solutionswith glucose concentrations of 20 mg/dl and 200 mg/dl. It was removed atdifferent times and placed in a NMR tube. T2 values were obtained inless than 30 seconds and the device placed in the original glucosesolution of a glucose solution with a different concentration.

Data processing: The line drawn for cyclical changes in T2 was obtainedby use of the following equation: T2=A*sin(B*time+C)+Y, A=15.58,B=0.0253836, C=50.283 and Y=83.8099.

Example 1 Glu-CLIO Nanoswitches

Summary

To demonstrate the water relaxation sensor we designed a prototype formonitoring physiological concentrations of glucose. We employedconconavalin A as a binding protein and synthesized a glucosefunctionalized magnetic nanoparticle (Glu-CLIO). Conconavalin A (ConA)is a tetravalent lectin that is known to react with glucose. Some of thesensors were prepared having a walled enclosure with a pore size of 3kDa. In general, the walled enclosure of the sensors retained theGlu-CLIO nanoparticle and ConA, while permitting glucose to freely enteror leave the sensor.

Example 1A Preparation of Glu-CLIO

MION47 and amino-CLIO (25-35 nm) were prepared as described elsewhere.D-Glucose, D-(+)-Glucosamine hydrochloride, succinic anhydride,Concanavalin A (ConA) and Sephadex G-25 were from Sigma Aldrich Co.1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) andsulfo-N-hydroxysuccinimide (sulfo-NHS) were from Pierce (Rockford,Ill.). To synthesize a glucose-functionalized nanoparticle (Glu-CLIO),NH₂-CLIO was first converted to a carboxylic group functionalizednanoparticle, followed by coupling of 2-amino-glucose using awater-soluble carbodiimide. To obtain a carboxylic functionalized CLIO,2.0 mg succinic anhydride was added into 200 uL NH₂-CLIO (10 mg Fe/mL,42 NH₂ per 2064 Fe) with 300 uL (0.1 M) NaHCO₃ buffer, pH 8.5. Themixture was incubated at room temperature for two hours and succinicacid removed using a Sephadex G-25 column eluted with MES buffer (0.5 MNaCl, 0.05 M MES), pH 6.0. To conjugate 2-amino-glucose to carboxyfunctionalized CLIO (CLIO-COOH), 2 mg EDC and 2 mg sulfo-NHS were addedinto 500 uL CLIO-COOH (mg Fe/mL) in MES buffer, pH 6.0. The mixture wasallowed to react for two hours at room temperature and purified bySephadex G-25 column eluted with PBS buffer at pH 7.4. Subsequently 2 mgGlucosamine was added into above solution and the mixture reacted forone hour at room temperature. Unreacted glucosamine was removed withSephadex G-25 in PBS.

Example 1B Glu-CLIO-ConA-Glucose Tube Assay

Relaxation times were obtained at 0.47T, 40° C. using a Minispecrelaxometer (Bruker).

To demonstrate the interaction between Glu-CLIO, ConA and glucose,experiments were performed directly in NMR tube (no semi-permeablewalled enclosure), 10 ug Fe/mL, 800 ug/mL ConA. All experiments wereperformed in PBS with 1 mM CaCl₂ and 1 mM MgCl₂. Transverse relaxationtimes (T2's) were measured in the relaxometer, Bruker Minispec® NMS 120at 0.47 T and 40° C. Size was determined with a Zetasizer HS1000®(Malvern Instruments, Marlboro, Mass.) in the buffer above with Glu-CLIOat 20 ug Fe/mL, followed by the addition of ConA to 1 mg/mL and 1.5mg/mL glucose. All experiments used these concentration of ions, buffer,ConA and Glu-CLIO.

As shown in FIG. 3A, the addition of ConA to Glu-CLIO resulted in a dropin T2 that reached a plateau after about 50 minutes, while no change wasobtained with amino-CLIO. Associated with the ConA induced T2 change wasan increase in the size of the Glu-CLIO nanoparticle from 30 nm to 301nm by laser light scattering, indicating nanoparticle clustering wasassociated with ConA addition and the T2 increase, and that the systemwas behaving like a magnetic relaxation switch. Addition of glucose thencaused a partial reversibility of the T2 drop, with T2 values againreaching a plateau after about 100 minutes, 150 minutes, and 200minutes. This type of response was seen with additions that producedconcentrations of 0.4, 0.8 and 1.8 mg/mL glucose. The sensor respondedto 0.2 to 1.8 mg/mL glucose, which is approximately the physiologicalrange of plasma glucose in humans. The apparatus is shown in FIG. 3B.

Example 1C Glu-CLIO-ConA-Glucose Tube Assay

Glu-CLIO nanoswitches were diluted into tubes to obtain a T2 of 153msec. Upon addition of ConA, T2 decreased and reached a plateau value of65 msec (see FIG. 4A).

Addition of increasing concentrations of glucose reversed this effect,with a constant T2 value observed at each glucose concentration (seeFIG. 4B). The change in plateau T2 values occurred in a linear fashionover the physiological range of glucose concentrations (see FIG. 4B).

To further investigate the interconversion (switch) between the initialdispersed nanoparticle state and the microaggregated state, lightscattering measurements were obtained (see FIGS. 4C, 4D, and 4E).Dispersed nanoswitches had a mean diameter of 26 nm (see FIG. 4C), whichincreased to a mean diameter of 230 nm upon addition of ConA (see FIG.4D), and which returned to its original size distribution with theaddition of glucose (400 mg/dl) (see FIG. 4E). The initial T2 value of153 msec (see FIG. 4A) was not achieved upon addition of the glucose.The discrepancy between the initial and final T2 value is believed to bedue to a slight dilution of iron that accompanied the addition ofconcentrated solutions of glucose. Based on the return of nanoswitchesto their original size distribution (see FIG. 4E), a completeinterconversion between dispersed and microaggregated nanoswitch stateswas achieved by glucose addition.

Example 1D Glu-CLIO-ConA-Glucose Sensor Assay

We next placed the components of the tube-based relaxation assay (ConAand Glu-CLIO as used in FIGS. 3A and 3B) in the semi-permeable deviceshown in FIG. 5B, to obtain a water relaxation based sensor (volume ˜0.5mL, 3 kDa pores). We allowed the sensor to equilibrate with 0.1 mg/mLglucose overnight and obtained a stable T2 of 98 msec (see FIG. 5A),obtained by placing it in a glass tube used with the MR relaxometer. Thesensor was then placed in a second tube (1 mg/mL glucose) and T2 valuesmonitored (see FIG. 5A). A new plateau of 70 msec was attached afterabout 100 minutes, indicating glucose-induced dispersion of the Glu-CLIOnanoparticles (see FIG. 5A). The sensor was then placed in a third tubewith 0.1 mg/mL glucose. T2 increased and returned to a plateau of T2again at 98 msec (see FIG. 5A). Thus, the water relaxation based glucosesensor uses a T 2 dependent equilibrium between ConA and Glu-CLIO tosense external glucose in a reversible fashion.

Similar results were obtained when Glu-CLIO and ConA were enclosed in asemi-permeable sensor to interact with glucose in the external sensorenvironment (FIG. 5B), using 500 uL of G-ConA and ConA as above wereplaced in a membrane of cellulose ester with a 1 kDa cutoff and diameterof 7.5 mm (Spectra/Por®, Fisher Scientific). The sensor was placed in 50mL tube with a magnetic stir at the bottom of tube for mixing. Atvarious times the external concentration of glucose was varied, and thesensor removed, placed in an NMR tube, and T2 determined as above(results not shown).

Example 1E Glu-CLIO-ConA-Glucose Sensor Assay with MRI Imaging

We placed Glu-CLIO-ConA sensors in two test tubes, one with 2 mg/mLglucose and without glucose and imaged the tubes using a clinical MRimager (see FIG. 6A). To demonstrate the ability of MRI to detect theinteraction between Glu-CLIO and ConA the semi-permeable membrane,Glu-CLIO (10 ug Fe/mL) and Con A (800 ug/mL) were placed in a 1 kDacutoff, 5 mm diameter semi-permeable tube (Spectra/Por IrradiatedDispodialyzer, Fisher) and the sensor placed in a 50 mL tube as above.After 2 hours, the stir bar was removed and images obtained on aclinical GE Signa 1.5 T unit. (Image size 256×192, field of view 7×14Cm, slice thickness 1.5 mm using a turbo spin echo pulse sequence, TR2500, TE65).

As shown in FIGS. 6B and 6C, the sensor in the high glucose environmenthad higher signal intensity (brighter image), reflecting nanoparticledissociation and a higher (longer) T2. Thus, the concentration ofexternal glucose altered the signal intensity of water within the sensorthat was evident on an MR image.

Example 1F Glu-CLIO-ConA-Glucose Sensor Assay with MRI Imaging

We further examined whether the nanoswitch/binding protein equilibriumwould be maintained if the nanoswitches and binding proteins wereenclosed in a semipermeable device, with pores that would allow analyteto enter but which would retain nanoparticles and binding protein. Thiswould allow analyte concentrations to be raised or lowered, depending onthe sensor environment (dialysate). A number of different units wereinvestigated as possible including Spectra/Por tubing, Slide-A-Lyzermicrocassettes and dialysis fibers. Spectra/Por tubing permitted therapid and repeated transfer of the sensor from a 100 mL beaker, whereglucose concentrations were cycled between 20 mg/dl and 400 mg/dl to anMR relaxometer, where T2 measurements were made in less than a minute.The T2 of sensor water changed in cycled between about 68 and 100 msecas it responded to changing concentrations of glucose (see FIG. 7). Bothincreases and decreases in glucose concentration resulted in alterationof the nanoswitch microaggregation state, evident by changes in T2,further demonstrating the equilibrium nature of nanoswitches.

Example 2 HA-CLIO Nanoswitches Example 2A Preparation of HA-CLIO

To synthesize hemagglutinin peptide-CLIO, a thiolated influenzahemagglutinin (HA) peptide with a C-terminal cysteine (YPYDVPDVAGGC) wassynthesized by using Fmoc chemistry on Rink amide resin (Calbiochem,NovaBiochem) and was purified by reverse phase HPLC. The molecularweight of HA was confirmed by MALDI-TOF. To attach HA to nanoparticle,amino-CLIO was first reacted with SPDP. After purification, 200 μL SPDPmodified CLIO (5.0 mg/mL Fe) in PBS buffer, pH 7.4 was mixed with 100 μLof HA (50 mM) in DMSO. Reaction proceeded for 2 hours at roomtemperature. The CLIO conjugate was separated by Sephadex G-25 columnand eluted with PBS buffer, pH 7.4. The number of peptides pernanoparticle was determined by the SPDP method. Nanoparticles had 25 HAper 2000 Fe and the size distribution shown in FIG. 3.

Example 2B HA-CLIO-anti-HA-HA Tube Assay

Relaxation times were obtained at 0.47T, 40° C. using a Minispecrelaxometer (Bruker).

Nanoswitches had similar properties when HA-CLIO replaced Glu-CLIO andantibody to HA (anti-HA) replaced ConA as shown in FIGS. 8A-8E. As shownin FIG. 8A, T2 dropped from 162 to 141 msec with the addition ofanti-HA. Plateau values of T2 changed over a range of HA concentrationsbetween 50 and 400 nM (FIG. 8B), which was about 80 fold lower than theconcentrations of glucose needed to change T2 (2.5 μM-20 μM, FIG. 4B).Again light scattering data indicated that the analyte (HA) was capableof essentially completely reversing microaggregate formation (see FIGS.8C, 8D, and 8E).

Example 3 FA-CLIO Nanoswitches Example 3A Preparation of FA-CLIO

To synthesize FA-CLIO, amino-CLIO in PBS buffer, pH 7.4 was firstexchanged with MES buffer (50 mM MES hydrate, 0.1 M NaCl), pH 6.0 andthe solution was concentrated to 5.0 mg/mL. Then 100 μL (50 mM) folicacid in DMSO was added to 200 μL amino-CLIO (5.0 mg/mL Fe) in MESsolution, pH 6.0. This was followed by the addition of excess EDC (0.96mg, 5 μmol) and sulfo-NHS (1.1 mg, 5 μmol) in 100 μL DMSO. Reactionproceeded at room temperature for 2 hours and the product was purifiedby Sephadex G-25 column and eluted with PBS buffer, pH 7.4. Attachmentof FA was quantified by the loss of amine groups using SPDP, see above,and was 33 FA per 2000 Fe with the size distribution shown in FIG. 9C.

Example 3B FA-CLIO-anti-FA-FA Tube Assay

Relaxation times were obtained at 0.47T, 40° C. using a Minispecrelaxometer (Bruker).

The properties of the nanoswitch system were examined with FA-CLIOnanoparticles and anti-FA as the binding protein. As shown in FIG. 9A,T2 dropped from 155 msec to 113 msec with anti-FA addition. The range orconcentrations of FA associated with changing T2 values are shown inFIG. 9B (5-20 nM) and was about 1000 fold lower than the concentrationsof glucose measured (2.5 μM-20 μM, FIG. 4B). Again, light scatteringdata indicated that the analyte (HA) was capable of essentiallycompletely reversing microaggregate formation (see FIGS. 9C, 9D, and9E).

We conclude that surface functionalized nanoparticles and bindingproteins maintained an equilibrium, continuously switching between adispersed (disaggregated), low T2 state (20-40 nm) and microaggregatedhigh T2 state (200-250 nm), depending the concentration of exogenousanalyte. Based on light scattering data, high concentrations ofexogenous analytes completely reversed microaggregate formation,returning the system to its original dispersed state expected for anequilibrium process. As indicated by the use of nanoswitches and bindingproteins for glucose, FA, and HA, nanoswitches were able to detectchemically diverse analytes over a relatively wide range ofconcentration.

Other Embodiments

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention. Forexample, two or more chelating moieties can be incorporated into asingle monomeric substrate molecule. Accordingly, other embodiments arewithin the scope of the following claims.

1. A water relaxation-based sensor for detecting the presence of ananalyte in a sample, the sensor comprising: (i) a walled enclosureenveloping a chamber, wherein the wall comprises an opening for passageof the analyte into and out of the chamber; (ii) a plurality of magneticnanoparticles located within the chamber, each nanoparticle having atleast one moiety that is covalently or noncovalently linked to thenanoparticle; and optionally, (iii) at least one binding agent locatedwithin the chamber; wherein the opening is smaller in size than thenanoparticles, and is larger in size than the analyte; and wherein themoiety and the analyte each bind reversibly to the binding agent, whenpresent; or the analyte binds reversibly to the moiety.
 2. The sensor ofclaim 1, wherein the opening is smaller in size than the binding agent.3. The sensor of claim 1, wherein the wall comprises a plurality ofopenings for passage of the analyte into and out of the chamber, whereineach of the openings is smaller in size than the nanoparticles and thebinding agent, and each of the openings is larger in size than theanalyte.
 4. The sensor of claim 1, wherein the moiety comprises acarbohydrate, an antibody, an amino acid, a nucleic acid, anoligonucleotide, a therapeutic agent or a metabolite thereof, a peptide,or a protein.
 5. The sensor of claim 1, wherein the moiety comprises amolecular fragment of the analyte being detected or a molecular fragmentof a derivative, isostere, or mimic of the analyte being detected. 6.The sensor of claim 1, wherein the moiety is linked to the nanoparticleby a functional group comprising —NH—, —NHC(O)—, —(O)CNH—,—NHC(O)(CH₂)_(n)C(O), —(O)C(CH₂)_(n)C(O)NH—, —NHC(O)(CH₂)_(n)C(O)NH—,—C(O)O—, —OC(O)—, or —SS—, and wherein n is 0 to
 20. 7. The sensor ofclaim 6, wherein the functional group is —NHC(O)(CH₂)_(n)C(O)NH—.
 8. Thesensor of claim 7, wherein n is
 2. 9. The sensor of claim 1, wherein thebinding agent is absent.
 10. The sensor of claim 9, wherein the moietycomprises a protein.
 11. The sensor of claim 9, wherein: (a) when theanalyte is absent, the chamber comprises substantially disaggregatednanoparticles; and (b) when the analyte is present, the chambercomprises a nanoparticle aggregate, wherein the nanoparticle aggregatecomprises nanoparticles bound to the exogenous analyte through themoiety.
 12. The sensor of claim 1, wherein the binding agent is present.13. The sensor of claim 12, wherein the binding agent comprises aprotein or a monoclonal antibody.
 14. The sensor of claim 12, whereinthe moiety comprises a molecular fragment of the analyte being detectedor a molecular fragment of a derivative, isostere, or mimic of theanalyte being detected.
 15. The sensor of claim 12, wherein: (a) whenthe analyte is absent, the chamber comprises a nanoparticle aggregate,wherein the nanoparticle aggregate comprises nanoparticles bound to thebinding agent through the moiety; and (b) when the analyte is present,the nanoparticles are displaced from the binding agent by the analyte,and the chamber comprises substantially disaggregated nanoparticles. 16.The sensor of claim 1, wherein the opening has a size of from about 1kDa to about 3 kDa.
 17. The sensor of claim 1, wherein each of thenanoparticles has an overall size of from about 30 nm to about 60 nm.18. The sensor of claim 11 or 15, wherein the nanoparticle aggregate hasa an overall size of at least about 100 nm.
 19. The sensor of claim 11or 15, wherein the change in nanoparticle aggregation between (a) and(b) alters the proton relaxation of water inside of the chamber, butdoes not substantially alter the proton relaxation of water outside ofthe chamber.
 20. The sensor of claim 19, wherein the change innanoparticle aggregation between (a) and (b) produces a measurablechange in the T2 relaxation times of water inside the chamber.
 21. Thesensor of claim 1, wherein the moiety comprises a chiral compound. 22.The sensor of claim 1, wherein the moiety comprises a carbohydrate. 23.The sensor of claim 1, wherein the moiety comprises the structure:


24. The sensor of claim 1, wherein the binding agent is a protein thatcomprises at least two binding sites.
 25. The sensor of claim 1, whereinthe binding agent is a protein that comprises at least four bindingsites.
 26. The sensor of claim 1, wherein the binding agent is a proteinthat binds to a carbohydrate.
 27. The sensor of claim 26, wherein thecarbohydrate is glucose.
 28. The sensor of claim 27, wherein the proteinis conconavalin A.
 29. The sensor of claim 1, wherein the binding agentcomprises a monoclonal antibody, a polyclonal antibody, or aoligonucleotide.
 30. The sensor of claim 1, wherein the magneticnanoparticles each comprise a magnetic metal oxide.
 31. The sensor ofclaim 30, wherein the magnetic metal oxide comprises a superparamagneticmetal oxide.
 32. The sensor of claim 30, wherein the metal oxidecomprises iron oxide.
 33. The sensor of claim 32, wherein each of themagnetic nanoparticles is an amino-derivatized cross-linked iron oxidenanoparticle.
 34. The sensor of claim 1, wherein the nanoparticles aresubstantially aggregated.
 35. A method of detecting an analyte in anaqueous sample, the method comprising: (i) providing the sensor of claim1; (ii) measuring relaxation times of the water inside of the chamber ofthe sensor in the absence of the analyte or under conditions that mimicthe absence of the analyte; (iii) contacting the sensor with the sample;(iv) measuring relaxation times of the water inside of the chamber ofthe sensor; and (v) comparing the T2 relaxation times measured in step(ii) and step (iv); wherein a change in T2 relaxation times measured instep (iv) relative to the T2 relaxation times measured in step (ii)indicates the presence of the analyte.
 36. The method of claim 35,wherein the analyte is a monovalent analyte.
 37. The method of claim 35,wherein the analyte is a multivalent analyte.
 38. The method of claim35, wherein the change in the T2 relaxation times is measured using amagnetic resonance imaging method.
 39. The method of claim 35, whereinthe change in the T2 relaxation times is measured using a magneticresonance non-imaging method.
 40. The method of claim 35, wherein theanalyte is a carbohydrate.
 41. The method of claim 35, wherein theanalyte is glucose.
 42. The method of claim 35, wherein the analyte ischiral.
 43. The method of claim 42, wherein the chiral analyte ispresent together with one or more optically active moieties in thesample.
 44. The method of claim 43, wherein the chiral analyte ispresent together with a stereoisomer of the chiral analyte in thesample.
 45. The method of claim 44, wherein the chiral analyte ispresent together with an enantiomer of the chiral analyte in the sample.46. The method of claim 42, wherein the chiral analyte is an amino acid.47. The method of claim 35, wherein the analyte is a nucleic acid or anoligonucleotide.
 48. The method of claim 35, wherein the analyte is atherapeutic agent or a metabolite of a therapeutic agent.
 49. The methodof claim 35, wherein the analyte is peptide or a protein.
 50. The methodof claim 35, wherein steps (ii) and (iv) comprise measuring T2relaxation times.
 51. The method of claim 50, wherein an increase in T2relaxation times measured in step (iv) relative to the T2 relaxationtimes measured in step (ii) indicates the presence of the analyte.